OBJECTIVE: To study chondrogenesis of calcium alginate-chondrocytes predetermined shapes. METHODS: Chondrocytes isolated from ears of rabbit by type II collagenase digestion, and then were mixed with 1.5% solidium alginate solution. The suspension was gelled to create three spatial shapes as triangle, circle and quadrilateral by immersed into 2.5% CaCl2 for 90 minutes, and then was implanted into the subcutaneous pocket on the dorsum of the rabbit. Samples were harvested at 6 and 12 weeks after implantation. RESULTS: Gross examination of excised specimens at 6 and 12 weeks after implantation revealed the presence of new cartilage of approximately the same dimensions as the original construct. Histologic evaluation using hematoxylin and eosin stains confirmed the presence of cartilage nodules at 6 weeks after implantation. After 12 weeks, mature cartilage was observed and histologic analysis confirmed the presence of well formed cartilaginous matrix. CONCLUSION: Predetermined shapes neocartilage can be regenerated using calcium alginate as a carrier of chondrocytes in the bodies of immune animals.
Objective To evaluate the cytocompatibility of collagenmembraneswith transitional cells of rabbit in vitro and to discuss the possibility of the collagen membranes as urologic tissue engineering scaffolds. Methods Primary cultured transitional cells isolated from New Zealand rabbits were implantedon collagen membranes at 1×105 cells/cm2. The changes of cell adhering were observed by inverted microscope and scanning electron microscope 2, 12 and 24hours later. The experiment was divided into 4 groups: non-cell group (black control) culture medium group(negative control), extract medium from Polyvinyl chloride group(positive control) and extract medium from collagen membranes group(experimental group). The cells of generations 2 to 4 were implanted in 96-hole-plank at 1×104 cells every hole. And every group had 5 holes. Then absorption coefficient were detected at the wave length of 490 nm by MTT assay. Then the cytotoxicity and cytocompatibility were evaluated by comparison of the numbers of absorptioncoefficient.Results The bladder transitional cells began to adhere to the collagen membrane 2 hours after implanting, and the number of the adhered cells increased with time.The actual absorption coefficient of experimental groups was 0.590±0.024,1.065±0.040 and 1.129±0.074 after 24, 72 and 120 hours. The actual absorption coefficient of negative control group was 0.639±0.068,1.022±0.044 and 1.087±0.111. The actual absorption coefficient of positive control group was 0.302±0.029,0.653±0.083 and 0.694±0.031. There was significantdifference between the experimental group and positive control (Plt;0.01), and no significant difference between the experimental group and negative control(Pgt;0.05).Conclusion Collagen membrane has good cytocompatibility withtransitional cells and no cytotoxity. It can be used as scaffolds of urologic tissue engineering.
Objective To review the biochemical characteristics, appl ication progress, and prospects of the adiposederived stem cells (ADSCs). Methods The recent original experimental and cl inical l iterature about ADSCs was extensively reviewed and analyzed. Results ADSCs can be readily harvested in large numbers from adipose tissue with properties of stable prol iferation and potential differentiation in vitro. Significant progress of ADSCs is made in the animal experimentand the cl inical appl ication. It has been widely used in the cl inical treatment of cardiovascular disease, metabol ic disease, encephalopathy, and tissue engineering repair. Conclusion ADSCs have gradually replaced bone marrow mesenchymal stem cells and become the focused hot spot of regenerative medicine and stem cells.
Objective To investigate the possible mechanism of the fibroblasts inducing the vascularization of dermal substitute. Methods Fibroblasts were seeded on the surface of acellular dermal matrix and cultivated in vitro to construct the living dermal substitute. The release of interleukin 8 (IL 8) and transfonming growth factor β 1(TGF β 1) in culture supernatants were assayed by enzyme linked immunosorbent assay, the mRNA expression of acid fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) were detected by RT-PCR. Then, the living substtute was sutured to fullth ickness excised wound on BALBouml;C m ice, and the fate of fibroblast w as observed by using in situ hybridizat ion. Results Fibroblasts cultured on acellular dermalmat rix p ro liferated and reached a single2layer confluence. Fibroblasts could secret IL 28 (192. 3±15. 9) pgouml;m l and TGF-B1 (1. 105±0. 051) pgouml;m l. There w as the mRNA exparession of aFGF and bFGF. Fibroblasts still survived and proliferated 3 weeks after graft ing. Conclusion Pept ides secreted by fibroblasts and its survival after graft ing may be relat ive to the vascularizat ion of the dermal subst itute.
OBJECTIVE: To study the feasibility of the formation of allogeneic tissue-engineered cartilage of certain shape in immunocompetent animal using the injectable biomaterial. METHODS: Fresh newborn rabbits’ articular cartilages were obtained under sterile condition (lt; 6 hours after death) and incubated in the sterile 0.3% type II collagenase solution. After digestion of 8 to 12 hours, the solution was filtered through a 150 micron nylon mesh and centrifuged, then the chondrocytes were washed twice with phosphate buffered saline (PBS) and mixed with the biomaterial to create a final cell density of 5 x 107/ml. The cell-biomaterial admixture was injected into rabbits subcutaneously 0.3 ml each point while we drew the needle back in order to form the neocartilage in the shape of cudgel, and the control groups were injected with only the biomaterial or the suspension of chondrocytes with the density of 5 x 10(7)/ml. After 4, 6, 8 and 12 weeks, the neocartilages were harvested to analyze. RESULTS: The new nodes could be touched subcutaneously after 2 weeks. In the sections of the samples harvested after 4 weeks, it was found that the matrix secreted and the collagen formed. After 6 weeks and later than that, the neocartilages were mature and the biomaterial was almost completely degraded. The cudgel-shaped samples of neocartilage could be formed by injection. In the experiment group, there was no obvious immune rejection response. On the contrary, there were no neocartilage formed in the control group. CONCLUSION: The injectable biomaterial is a relatively ideal biomaterial for tissue engineering, and it is feasible to form allogeneic tissue engineered cartilage of certain shape by injection in an immunocompetent animal.
Objective To evaluate the biocompatibility of a new bone matrix material (NBM) composed of both organic and inorganic materials for bone tissue engineering. Methods Osteoblasts combined with NBM in vitro were cultured. The morphological characteristics was observed; cell proliferation, protein content and basic alkaline phosphatase(ALP) activity were measured. NBM combined with osteoblasts were implanted into the skeletal muscles of rabbits and the osteogenic potential of NBM was evaluated through contraat microscope, scanning electromicroscope and histological examination. In vitro osteoblasts could attach and proliferate well in the NBM, secreting lots of extracellular matrix; NBM did not cause the inhibition of proliferation and ALP activity of osteoblasts. While in vivo experiment of the NBM with osteoblasts showed that a large number of lymphacytes and phagocytes invading into the inner of the material in the rabbit skeletalmuscle were seen after 4 weeks of implantation and that no new bone formation was observed after 8 weeks. Conclusion This biocompat ibility difference between in vitro and in vivo may be due to the immunogenity of NBM which causes cellular immuno reaction so as to destroy the osteogenic environment. The immunoreaction between the host and the organic-inorganic composite materials in tissue engineering should be paid more attention to.
OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro. METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days. Human satellite cells were identified with monoclonal antibody against desmin. Cells were observed under phase contrast microscopy. RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium. After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours. CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition. Immunocytochemical detection of desmin is the effective early method to determine satellite cell.
Objective To comment on the recent advances of production and application of the bio-derived scaffold in the tissue engineered peripheral nerve. Methods The recent articles were systematically analyzed, and then the production methods of the bio-derived scaffold and its application to the tissue engineered peripheral nerve were evaluated and prospected. Results B iological tissues were processed by some methods to produce the bio-derived materials. These mat erials could maintain the structure and components of the tissues. Moreover, the immunogenicity of these materials was reduced. Conclusion Application of the bio-derived materials is a trend in the fabricating scaffold of the tissue en gineered peripheral nerve.
OBJECTIVE This paper aims to investigate the suitable cell density and the best formation time of tissue engineered autologous cartilage and to provide theoretical basis and parameters for clinical application. METHODS The chondrocytes isolated from mini swines’ ears were mixed with injectable biocompatible matrix (Pluronic), and the density of cell suspensions were 10, 20, 30, 40, 50, 60, 70 x 10(4)/ml. The chondrocyte-polymer constructs were subcutaneously injected into the abdomen of autologous swine. The specimens were observed grossly and histologically after 6 weeks, and investigated the suitable cell density. Then the chondrocyte-polymer constructs with suitable cell density were transplanted into the abdomen of autologous swine and evaluated grossly and histologically in 1, 3, 6, 9, 15 weeks after transplantation to investigate the best formation time of tissue engineered cartilage. RESULTS The experiments demonstrated that the tissue engineered autologous cartilage was similar to the natural cartilage on animals with normal immune system in histological characteristics. The optimal chondrocyte density is 50 x 10(6)/ml, and the proper harvest time is the sixth week. CONCLUSION With tissue engineering skills, we have identified the optimal chondrocyte density and the proper harvest time.
OBJECTIVE To search an ideal carrier of transferred keratinocytes for transplantation. METHODS The transferred keratinocytes were seeded on the surfaces of the artificial dermis and the silicone membrane and cultured in vitro for 2 weeks. The growth of the keratinocytes was observed by microscope and scanning electron microscope. RESULTS The keratinocytes implanted on the artificial dermis began to rupture and died after 2 to 3 days. While the keratinocytes adhered well on the surface of silicone membrane with pseudopodia formation after 1 week under scanning electron microscope, and the cells kept normal morphological and proliferative properties 2 weeks later. CONCLUSION The silicone membrane can be applied as an useful carrier for the keratinocytes transplantation.
